Prolonged release microcapsules

ABSTRACT

This invention provides a microcapsule designed for zero order release of a physiologically active polypeptide over a period of at least two months, which is produced by preparing a water-in-oil emulsion comprising an inner aqueous layer containing about 20 to 70% (w/w) of said polypeptide and an oil layer containing a copolymer or homopolymer having a weight-average molecular weight of 7,000 to 30,000, wherein the composition ratio of lactic acid/glycolic acid in the copolymer or homopolymer is 80/10 to 100/0, and then subjecting said water-in oil emulsion to microencapsulation.

This application is a division of application Ser. No. 08/458,679, filedJun. 2, 1995, now U.S. Pat. No. 5,643,607, which is a divisional ofapplication Ser. No. 08/188,918, filed Jan. 31, 1994, now U.S. Pat. No.5,480,656, which is a continuation of application Ser. No. 07/649,727,filed Feb. 1, 1991, now abandoned.

FIELD OF THE INVENTION

This invention relates to microcapsules designed for sustained releaseof physiologically active peptide.

BACKGROUND OF THE INVENTION

Various dosage forms have been proposed for drugs required to beadministered for a prolonged period. Among them, Japanese publishedunexamined patent application (Toku-Kai Sho) 57-118512 and itscorresponding EP-A-0052510 disclose preparation of microcapsules by aphase separation method using a coacervation agent such as a mineral oilor a vegetable oil. Toku-Kai Sho 60-100516 (the corresponding U.S. Pat.Nos. 4652441 and 4711782), 62-201816 (the corresponding EP-A-0190833)and 63-41416 disclose methods of preparing microcapsules by means ofin-water drying. According to these methods, drugs can be efficientlyincorporated into microcapsules to give desirable microcapsules withless initial release.

In the case of administering a drug in the form of microcapsules,requirements for microcapsules having high dependency on interactionwith functions of the living body are diversified into a variety ofphases. Since the matter is concerned with medicines, microcapsulescapable of satisfying those various requirements as far as possible havebeen desired.

There are many reports on microcapsules comprising a water-soluble drugusing a biodegradable polymer. However, in the case of using awater-soluble drug, especially a physiologically active peptide having arelatively large molecular weight, the diffusion of the drug thusencapsulated into the polymer is low, and, therefore, the drug is notreleased at the initial stage until the decomposition or impregnating ofthe polymer proceeds. Also, as an adverse effect, a large burst at theinitial stage cannot be avoided depending on the method of preparation.Thus it is often the case that practical difficulties occur with use asmedicines. Especially, in sustained release pharmaceutical compositionsover an extended period of time, constant release of the drug withhigher accuracy is an important requirement, but no microcapsulessatisfying those requirements have been known.

OBJECTS OF THE INVENTION

In view of these circumstances, the present inventors have conductedintensive studies with the purpose of developing pharmaceuticalcompositions designed for sustained release of a physiologically activepeptide over an extended period of time. As a result, the presentinventors found that, by preparing microcapsules using suitably selectedpolylactic acid of a limited molecular weight or lactic acid-glycolicacid (100/0 to 80/20), microcapsules having continuous excellentreleasability for a long time were obtained. Further research work basedon this finding has now led to completion of the present invention.

More specifically, the main object of the present invention is toprovide a microcapsule designed for zero order release of aphysiologically active polypeptide over a period of at least two months,which is produced by preparing a water-in-oil emulsion comprising aninner aqueous phase containing about 20 to 70% (W/W) of said polypeptideand an oil phase containing a copolymer or homopolymer having aweight-average molecular weight of 7,000 to 30,000, the compositionratio of lactic acid/glycolic acid being 80/10 to 100/0, and thensubjecting said water-in-oil emulsion to microencapsulation.

SUMMARY OF THE INVENTION

According to the present invention, there is provided the microcapsulesdesigned for zero order release of a physiologically active polypeptideover a period of at least two months.

DETAILED DESCRIPTION OF THE INVENTION

The physiologically active peptides usable in practice of this inventioninclude those comprising with two or more amino acid residues and havinga molecular weight of about 200 to about 100,000.

Examples of said peptides include luteinizing hormone-releasing hormone(LH-RH) and its analogs, for example, substances having LH-RH likeactivity cf. U.S. Pat. Nos. 3,853,837, 4,008,209, 3,972,859 and4,234,571, British Patent No. 1,423,083, Proceedings of the NationalAcademy of Sciences of the United States of America, Volume 78, pages6509-6512 (1981)! and LH-RH antagonists (cf. U.S. Pat. Nos. 4,086,219,4,124,577, 4,253,997 and 4,317,815). There may be further mentionedprolactin, adrenocorticatropic hormone (ACTH), melanocyte-stimulatinghormone (MSH), thyrotropin-releasing hormone (TRH), salts andderivatives thereof (cf. Toku-Kai Sho 50-121273, 51-116465),thyroid-stimulating hormone (TSH), lutenizing hormone (LH),follicle-stimulating hormone (FSH), vasopressin, vasopressin derivatives(desmopressin, etc.), oxytocin, calcitonin, parathyroid hormone (PTH)and its derivatives (cf. Toku-Kai Sho 62-28799), glucagon, gastrin,vasoactive intestinal peptide (VIP), lipocortin, vasocortin, atrialnatriuretic peptide (ANP), endothelin, secretin, pancreozymin,cholecystokinin, angiotensin, human placental lactogen, human chorionicgonadotropin (HCG), enkephalin, enkephalin derivatives cf. U.S. Pat. No.4,382,923, E. P. Appln. Pub. No. 31,567!, endorphin, kyotorphin,insulin, somatostatin, somatostatin derivatives (cf. U.S. Pat. Nos.4,087,390, 4,093,574, 4,100,117 and 4253,998), growth hormones, andvarious cell proliferation differentiation factors e.g. insulin-likegrowth factor (IGF), epidermal growth factor (EGF), fibroblast growthfactor (FGF), platelet-derived growth factor (PDGF), nerve growth factor(NGF), hepatic cell growth factor (HGF), transformed growth factor(TGF-β), bone morphogenetic factor (BMF), vascularization factor,vascularization inhibiting factor, fibronectin, laminine, etc.!,interferons (α-, β- and τ-type), interleukins (I, II, III, IV, V, VI andVII), tuftsin, thymopoietin, thymosin, thymostimulin, thymic humoralfactor (THF), serum thymic factor (FTS) and derivatives thereof (cf.U.S. Pat. No. 4,229,438), and other thymic factors cf. Proc. Natl. Acad.Sci. U.S.A., Vol. 78, pages 1162-1166 (1984)!, tumor necrosis factor(TNF), colony stimulating factor (CSF), motilin, erythropoietin (EPO),dynorphin, bombesin, neurotensin, cerulein, bradykinin, urokinase,prourokinase, tissue plasminogen activator (t-PA), and derivativesthereof (cf. "Therapeutic Peptides and Proteins". Cold Spring HarborLaboratory, New York, pp. 69-74, 1989), streptokinase, asparaginase,kallikrein, substance P., blood coagulation factors VIII and IX,lysozyme chloride, polymixin B, colistin, gramicidin, bacitracin, etc.

Especially, in a microcapsule comprising, as the physiologically activepolypeptide, an analog of LH-RH, which is water-solube and has amolecular weight of 1,000 or more, e.g. TAP-144 expressed by(pyr)Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHC₂ H₅, or LHRH antagonistexpressed by (pyr)Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-GlyNHC₂ H₅ !,continuous sustained release is performed advantageously over aprolonged period of time.

These physiologically active peptides are used in amounts selectedlargely depending on the kind of peptide, desired pharmacologicaleffects and duration of the effects, among others, and the amount rangesfrom about 0.01 mg to 5 g, more preferably, from 0.1 mg to 2 g, as thedosage of microcapsules. The concentration in a microcapsule depends onthe physico-chemical properties of the drug, and it is selected withinthe range of about 0.01% to about 50% (w/w), more preferably within therange of 0.1% to 30% (w/w).

The concentration of said polypeptide in the inner aqueous phase of amicrocapsule ranges from about 20% to 70% (w/w), preferably 25 to 65%(w/w), more preferably 35 to 60% (w/w) while it depends on itsphysico-chemical properties such as the solubility in water.

Examples of the polymer used as a release-controlling substance includecopolymers or homopolymers of lactic acid/glycolic acid which have anacid residue in the molecule, are hardly soluble or insoluble in waterand are biocompatible. The ratio of lactic acid/glycolic acid depends onthe period required for sustained release, and is selected from therange of 100/0 to 80/20, preferably 100/0 to 90/10, more preferably100/0.

As lactic acid, L-, D-and DL-lactic acid can be used, especially acopolymer or homopolymer prepared by polymerization of the monomer oroligomer of DL-lactic acid is advantageously utilized.

As the copolymer or homopolymer consisting of DL-lactic acid/glycolicacid, such polymers containing substantially no catalyst as obtained bypolymerization in the absence of catalyst are advantageously used (cf.Toku-Kai Sho 61-285215). Polymers having a dispersion degree (ratio ofweight-average molecular weight to number-average molecular weight) of1.5 to 3.0, especially 1.5 to 2.5 are preferable.

The length of the period of continuous sustained release ofmicrocapsules of this invention largely depends on the molecular weightof a polymer and the composition ratio of lactic acid/glycolic acid. Inthe case of preparing, for example, microcapsules performing continuouszero order release for at least three months, when the composition ratioof lactic acid/glycolic acid is 100/0, the preferable average-weightmolecular weight of a polymer ranges from 7,000 to 25,000; when 90/10,from 7,000 to 30,000; and when 80/20, from 12,000 to 30,000.

In the present specification, the weight-average molecular weight andthe degree of dispersion mean values which are determined by means of agel-permeation chromatography using commercially available polystyreneof standard molecular weight.

The concentration of a polymer in the oil phase when preparingmicrocapsules is selected from the range of about 0.5 to 90% (w/w), morepreferably from the range of about 2 to 60% (w/w).

The solution (oil phase) containing the above-mentioned polymer is thatof the polymer dissolved in an organic solvent.

Said organic solvent may be any organic solvent which has a boilingpoint not higher than about 120° C. and hardly miscible with water.Examples are halogenated alkanes (e.g. dichloromethane, chloroform,chloroethane, trichloroethane, carbon tetrachloride, etc.), ethylacetate, ethyl ether, benzene, toluene, etc. These may be used inadmixture of two or more.

In the present invention, desirable microcapsules showing less initialrelease can be prepared without adding a drug retaining substance, butsaid retaining substance may be supplemented according to the situation.The drug retaining substance mentioned above is a compound which givesincreased viscosity to the inner aqueous phase or solidifies by theaction of temperature, addition of ions, or a compound having a basicresidual group having protonic charge, which has interaction with apolymer to increase the viscosity of a W/O emulsion.

Examples of said drug retaining substance include gelatin, agar, alginicacid, polyvinyl alcohol, or a basic amino acid such as arginine, lysine,etc., a polypeptide containing a basic amino acid, an organic base suchas N-methyl glucamine, and a natural or synthetic basic polymer.

These compounds can be used singly or as a mixture of two or more ofthem. While the amount of these compounds to be used depends on theirkinds, it is preferable to have the concentration in the inner aqueousphase selected from the amount ranging from about 0.05% to 90% (w/w),more preferably from about 0.1% to 80% (w/w).

As conventional methods of controlling the releasability of thesemicrocapsules, mention is made of a method of changing the hydrolysisrate Biomaterials Vol. 5, 237-240 (1984)! and a method comprisingincorporation of a water-soluble compound into matrix of microcapsulesto create aqueous channels for releasing the drug. However, the formertends to invite shortening of a long-term of release, and the latterinduces only an initial burst, thus an approximate zero-order releasecan hardly be expected, Chem. Pharm. Bull. Vol. 36(4) 1502-1507 (1988)!.And, in the latter case, there is a fear of occurrence of undesirableside effects due to the increase of drug in blood at the initial stage.Further, there is also a known method (Toku-Kai Sho 57-150609), whichcomprises having the polymerization ratio of lactic acid/glycolic acidof PLGA to improve the time of suspending the release. This method is,however, directed to increase the speed of decomposition of the polymer,which, naturally, shortens the period of the release, thus there is alimit in realizing continuous release for a long period of time.

The sustained-release microcapsules of the present invention areprepared by, for example, the following method.

Stating more concretely, first, a physiologically active peptide isadded to water in an amount required for realizing the above-mentionedconcentration, to which is further added, when necessary, adrug-retaining substance such as the above-mentioned gelatin or basicamino acid to make a solution or a suspension having the above-mentionedconcentration, to prepare the inner aqueous phase.

To this inner aqueous phase, there may be added a pH-adjusting agent formaintaining the stability or solubility of the physiologically activepeptide, such as carbonic acid, acetic acid, oxalic acid, citric acid,phosphoric acid, hydrochloric acid, sodium hydroxide, arginine, lysineand their salts. And, there may further be added, as a stabilizer of thephysiologically active peptide, albumin, gelatin, citric acid, sodiumethylenediamine tetraacetate, dextrin, sodium hydrogen sulfite or apolyol compound such as polyethylene glycol, or, as a preservative,there may be added conventionally usable ones, such as apara-hydroxybenzoic acid ester (e.g. methylparaben, propylparaben),benzyl alcohol, chlorobutanol or thimerosal.

The thus-obtained inner aqueous phase is added to a polymer-containingsolution (oil phase), followed by an emulsification procedure to give aW/O type emulsion.

For said emulsification procedure, a known method of effectingdispersion is employed. As the method, mention is made of, for example,the intermittent shaking method, the method using a mixer such as apropeller-shaped stirrer, a turbine-shaped stirrer or the like, thecolloid mill method, the homogenizer method or the ultrasonificationmethod.

Then, the thus-prepared W/O emulsion is subjected to microencapsulation.An in-water drying or phase-separation method may be employed as a meansof microencapsulation. In the case of preparing microcapsules by thein-water drying, said W/O emulsion is further added to a third aqueousphase to give a W/O/W ternary emulsion and, thereafter, the solvent inthe oil phase is evaporated off to give microcapsules.

To the external aqueous phase, there may be added an emulsifying agent.As the emulsifying agent, there may be used any one capable of forminggenerally a stable O/W emulsion, for example an anionic surfactant (e.g.sodium oleate, sodium stearate, sodium lauryl sulfate, etc.), a nonionicsurfactant (e.g. polyoxyethylenesorbitan fatty acid ester (Tween 80,Tween 60, products of Atlas Powder Co.), a polyoxyethylene castor oilderivative (HCO-60, HCO-50, products of Nikko Chemicals), etc.),polyvinyl pyrrolidone, polyvinyl alcohol, carboxymethylcellulose,lecithin or gelatin. Such emulsifiers may be used either alone or incombination. The emulsifying agent concentration may suitably beselected within the range of about 0.01% to 20%, preferably within therange of about 0.05% to 10%.

For evaporation of the solvent from the oil phase, any of the commonmethods in general use can be employed. The method is conducted by, forexample, gradually reducing the pressure while stirring with apropeller-shaped stirrer or a magnetic stirrer, or by using a rotaryevaporator while adjusting the degree of vacuum. In this case, therequired time can be reduced by gradually warming the W/O/W emulsionafter the progress of solidification of the polymer to a certain extentfor rendering the solvent removal more complete.

The thus-produced microcapsules are collected by centrifugation orfiltration, rinsed several times with distilled water to thereby removethe free physiologically active peptide, drug retaining substance andthe emulsifying agent adhering to the microcapsule surface, followed bydispersing the resultant microcapsules in e.g. distilled water and byfreeze-drying, which is, if necessary, warmed under reduced pressure tothereby remove the moisture in the microcapsules and the solvent in themicrocapsule wall more completely.

In the case of preparing microcapsules by the phase-separation method, acoacervation agent is gradually added to the said W/O emulsion understirring to allow the polymer to precipitate and solidify.

A coacervation agent may be any solvent-miscible polymeric, mineral oilor vegetable oil compounds as exemplified by silicone oil, sesame oil,soybean oil, corn oil, cotton seed oil, coconut oil, linseed oil,mineral oils, n-hetane, n-heptane etc. These may be used as a mixture oftwo or more of them.

The microcapsules obtained thus above were collected by filtration andwashed with, for example, heptane, repeatedly to remove the solvent ofthe polymer. Further, removal of the free drug and separation of thesolvent were conducted in a manner similar to the in-water dryingprocess. For preventing aggregation of microcapsules to one anotherduring the washing, an agent for preventing aggregation may be added.

The microcapsules of the present invention designed forsustained-release produced by the above-mentioned in-water dryingprocess more preferably perform a stable sustained-release for a longperiod of time.

Dosage forms of administering microcapsules for the present inventioninclude injections, implantations and agents absorbed through mucousmembrane of rectum or uterus.

The microcapsules obtained in the above manner are sieved, whennecessary after slightly crushing, to eliminate excessively largemicrocapsules. The average grain size of microcapsules is within therange from about 0.5 to 1000 μm, desirably and preferably within therange of about 2 to 500 μm. When the microcapsules are used asinjections in the form of suspension, the grain size may be sufficientso long as it satisfies the requirements for dispersability andinjectability, for example, desirably within the range of about 2 to 100μm.

The microcapsules produced by the methods according to this inventionhave many advantages. For instance, they scarcely undergo aggregation orcohesion to one another during the production step. There can beobtained microcapsules which are satisfactorily spherical in shapehaving an optional size. The step of removing the solvent from the oilphase is easy to control, whereby the surface structure ofmicrocapsules, which is decisive for the rate of drug release can becontrolled.

The microcapsules produced by the method of this invention can be easilyadministered as injections and implants intramuscularly, subcutaneously,intravenously, or at an organ, joint cavity or at a lesion such astumors. They may also be administered in various dosage forms and thuscan be used as materials in preparing such dosage forms.

For instance, in making up the microcapsules according to this inventionfor an injection, the microcapsules according to the invention aredispersed in an aqueous medium together with a dispersing agent (e.g.Tween 80, HCO-60, carboxymethylcellulose, sodium alginate, etc.), apreservative (e.g. methylparaben, propylparaben, etc.), an isotonizingagent (e.g. sodium chloride, mannitol, sorbitol, glucose, etc.), orsuspended in an aqueous medium together with a vegetable oil such assesame oil or corn oil. Such dispersion or suspension is formulated intoa practically usable sustained-release injection.

Furthermore, the above microencapsulated sustained-release injection canbe converted to a more stable, sustained-release injection by adding anadditional excipient (e.g. mannitol, sorbitol, lactose, glucose, etc.),redispersing the resulting mixture and effecting solidification byfreeze-drying or spray drying with extemporaneous addition of distilledwater for injection or some appropriate dispersing agent.

The dose of the sustained-release preparation according to thisinvention may vary depending on the kind and amount of thephysiologically active peptide, which is the active ingredient, dosageform, duration or drug release, recipient animal (e.g. warm-bloodedanimals such as mouse, rat, rabbit, sheep, pig, cow, horse, human) andpurpose of administration but should be within the range of effectivedose of said active ingredient. For example, the single dose per saidanimal of the microcapsules can adequately be selected within the rangeof about 0.1 mg to 100 mg/kg body weight, preferably about 0.2 mg to 50mg/kg body weight.

In this manner, there is obtained a pharmaceutical composition preparedin the form of microcapsules which comprises an effective but greateramount of the physiologically active peptide as compared with theordinary single dose and a biocompatible polymer and is capable ofreleasing the drug continuously over a prolonged period of time.

The sustained-release preparation according to the present invention hasthe following characteristics, among others: (1) Continuoussustained-release of the physiologically active peptide can be attainedin various dosage forms. In particular, where a long-term treatment withan injection is required, the desired therapeutic effects can beachieved in a stable manner by injection of the preparation once inthree month, or once in six months, instead of daily administration.Thus, said preparation can achieve a sustained drug release over alonger period as compared with the conventional sustained-releasepreparations. (2) When the preparation in which a biodegradable polymeris used is administered in the form of an injection, such surgicaloperation as implantation is no more required but the preparation can beadministered subcutaneously, intramuscularly, or at an organ or alesion, with ease in quite the same manner as the ordinary suspensioninjections. And, there is no need for taking the matrix out from thebody after completion of the drug release.

The following Reference Example and Examples illustrate the invention infurther detail.

REFERENCE EXAMPLE 1

A four-necked flask equipped with a thermometer, a condenser and aninlet of nitrogen was charged with 160 g of a 85% aqueous solution ofDL-lactic acid. The solution was heated under reduced pressure for sixhours in nitrogen streams at inner temperatures and pressures rangingfrom 105° C. and 350 mmHg to 150° C. and 30 mmHg to remove water thusdistilled. The reaction was allowed to proceed at 175° C. for 90 hoursunder reduced pressure of 3 to 5 mmHg, and was then cooled to roomtemperatures to give 98 g of a substantially colorless massive polymer.This polymer was dissolved in tetrahydrofuran and the weight-averagemolecular weight and the degree of dispersion were determined by meansof a gel-permeation chromatography using commercially availablepolystyrene of standard molecular weight to find 17,200 and 1.89,respectively.

EXAMPLE 1

TAP-144 (400 mg) was dissolved in 0.5 ml of distilled water to give anaqueous phase. The aqueous solution was added to a solution of 4 g ofpoly-DL-lactic acid Lot No. 870818, weight-average molecular weight18,000 (microcapsule Lot No. 244, 245) and Lot No. 880622,weight-average molecular weight 18,200, dispersity 1.76 (microcapsuleLot No. 248)! in 7.5 ml of dichloromethane. The mixture was stirred in asmall-size homogenizer (Polytron, product of Kinematica, Switzerland)for about 60 seconds to give a W/O emulsion. This emulsion was cooled to15° C. This emulsion was then poured into 1,000 ml of a 0.25% aqueoussolution (previously cooled at 15° C.) of polyvinyl alcohol (PVA) whichwas stirred using a small-size homogenizer to give a W/O/W emulsion.Thereafter, dichloromethane was evaporated off, while stirring the W/O/Wemulsion, to thereby solidify the inner W/O emulsion, followed bycollecting thus solidified material by centrifugation.

The material was again dispersed in distilled water, which was subjectedto centrifugation, followed by washing the drug and the dispersant thenliberated.

Microcapsules thus collected were subjected to freeze-drying to removethe solvent and to dehydrate more completely to give powdery product.The content of the drug to be taken up in the microcapsules (Lot. 244,245, 248) was prescribed as 9%, and the entrapped ratio was 100% ormore.

These microcapsules were administered to rats (n=5) subcutaneously, thenthe TAP-144 remaining in the microcapsules at the injection site wasdetermined quantitatively to measure the in vivo release rate of thedrug. The results are shown in Table-1.

                  TABLE 1                                                         ______________________________________                                        in-vivo release-rate                                                                 Amount of Drug Remaining Subcutaneously (%)                            Lot    1 day    2 weeks 4 weeks 8 weeks                                                                             14 weeks                                ______________________________________                                        244    102.2    89.0    70.2    44.0  9.5                                     245    105.9    82.4    69.4    52.1  9.8                                     248    104.1    75.4    72.8    43.7  11.6                                    ______________________________________                                    

These microcapsules do not show initial burst and continuous release ofTAP-144 was observed for 14 weeks, i.e. longer than 3 months, withsubstantially good reproducibility.

EXAMPLE 2

Similarly, TAP-144 (400 mg) was dissolved in 0.5 ml of distilled waterto give an aqueous phase. Four grams of poly-DL-lactic acid having aweight-average molecular weight of 8,400 (Lot. 870304, microcapsule Lot.312) was dissolved in 5 ml of dichloromethane to give an oil phase. Theaqueous phase and the oil phase were mixed in the same manner asdescribed above to give a W/O emulsion.

This emulsion was cooled to 13° C., and then poured into 1,000 ml of0.25% aqueous solution of polyvinyl alcohol (PVA). The mixture wasprocessed in the same manner as described above to give a W/O/Wemulsion, which was prepared into microcapsules.

Further, 550 mg of TAP-144 was dissolved in 1 ml of distilled water. Onthe other hand, 4 g each of three samples of poly-DL-lactic acid (LotNo. 890717, molecular weight 14,100, dispersity 2.00 microcapsule Lot.402; Lot No. 890720, molecular weight 17,200, dispersity 1.89,microcapsule Lot No. 405; Lot No. 890721, molecular weight: 17,500,dispersity: 1.87, microcapsule Lot No. 406) was dissolved in 7.5 ml eachof dichloromethane. The above aqueous solution was added to each of thethree samples dissolved in dichloromethane, followed by processing inthe same manner as above to give three samples of W/O emulsion. Therespective emulsions were poured into 1,000 ml each of three samples of0.25% aqueous solution of polyvinyl alcohol previously cooled at 15° C.(the first one) and at 18° C. (the second and third ones), which wererespectively processed in the same manner as described in the foregoingto obtain microcapsules. The entrapped ratios of the drug were 101%,113% and 103%, respectively.

Table-2 shows in vivo release rates of the drug in the respectivemicrocapsules measured in the same manner as described above.

                  TABLE 2                                                         ______________________________________                                                    Amount of Drug Remaining Subcutaneously (%)                       Lot  n      1 day  1 week                                                                              2 weeks                                                                             8 weeks                                                                             12 weeks                                                                             14 weeks                          ______________________________________                                        312  5      86.3   82.2  41.2  9.8    --    --                                402  3      98.0   78.2  64.9  38.4   20.0  --                                405  5      88.8   79.4  52.2  33.8   --    21.3                              406  5      85.5   86.2  56.7  38.8   --    23.1                              ______________________________________                                    

The release of the drug, after a small amount of initial release, showsa continuous long release over longer than two months. The term ofrelease was dependent upon the hydrolysis rate of the high molecularpolymer then employed.

EXAMPLE 3

Microcapsules were prepared, in the same manner as in Example 1, from anaqueous phase prepared by dissolving 400 mg of TAP-144 in 0.5 ml ofdistilled water and an oil phase prepared by dissolving 4 g ofpolylactic acid-glycolic acid (90/10) Lot No. 870320 (weight-averagemolecular weight : 19,000), microcapsule Lot No. 315, Lot No.; 891020(weight-average molecular weight: 13,800), microcapsule Lot No. 410!.Referring to the microcapsule Lot No. 410, an aqueous solution preparedby dissolving 550 mg of TAP-144 in 1 ml of distilled water was used asthe inner aqueous phase, and the temperatures of the W/O emulsion, andthe external phase were adjusted to 15° C. and 18° C., respectively. Theentrapped ratios of the drug in these microcapsules were 106% and 100%,respectively.

These microcapsules were administered to rats subcutaneously in the samemanner as described above, and their in vivo release rates of the drugwere evaluated. Table-3 shows that sustained-release microcapsules for acontinuous prolonged period over more than two months were obtained.

                  TABLE 3                                                         ______________________________________                                             Amount of Drug Remaining Subcutaneously (%)                              Lot  1 day  1 week  2 weeks                                                                             4 weeks                                                                             6 weeks                                                                             8 weeks                                                                             10 weeks                          ______________________________________                                        315  77.4   76.0    59.2  51.6  41.1  25.8  --                                410  93.5   88.3    64.1  52.5  33.1  32.7  15.4                              ______________________________________                                    

EXAMPLE 4

Microcapsules were prepared, in the same manner as Example 1, from anaqueous phase prepared by dissolving 280 mg of TRH (free form) in 0.25ml of distilled water and an oil phase prepared by dissolving, in 6 mlof dichloromethane, poly-DL-lactic acid (average molecular weight17,200, dispersity 1.89) employed in Example 2, and by adjusting thetemperature of the W/O emulsion and external aqueous phase at 15° C. Theentrapped ratio of the drug in the microcapsules thus obtained (Lot No.R-103) was 85.8%.

Table-4 shows that the release of the drug in thus-obtained microcapsulewas as long-lasting as covering about 3 months.

                  TABLE 4                                                         ______________________________________                                               Amount of Drug Remaining Subcutaneously (%)                            Lot    1 day    2 weeks 4 weeks 8 weeks                                                                             12 weeks                                ______________________________________                                        R103   98.3     80.0    61.8    30.6  6.7                                     ______________________________________                                    

What we claim is:
 1. A microcapsule exhibiting zero order release ofluteinizing hormone-releasing hormone (LH-RH) analog for a period of atleast two months upon administration, which is produced by preparing awater-in-oil emulsion comprising an inner aqueous phase free from a drugretaining substance containing 35 to 60% (W/W) of the LH-RH analog, andan oil phase containing a homopolymer of lactic acid or a copolymer oflactic acid and glycolic acid with a ratio of lactic acid/glycolic acidof 100/0 to 90/10, the homo-or copolymer having a weight-averagemolecular weight of 13,800 to 19,000 and a dispersion degree (ratio ofweight-average molecular weight to number average molecular weight) of1.5 to 2.5, as the material for forming an outer wall of themicrocapsule; and then subjecting said water-in-oil emulsion tomicroencapsulation.
 2. A microcapsule according to claim 1, whichexhibits zero order release for a period of at least three months uponadministration.
 3. A microcapsule according to claim 1, wherein theconcentration of the copolymer or homopolymer in the oil phase is from 2to 60% by weight.
 4. A microcapsule according to claim 1, wherein theLH-RH analog is water-soluble and has a molecular weight of 1,000 ormore.
 5. A process for preparing a microcapsule exhibiting zero orderrelease of luteinizing hormone-releasing hormone (LH-RH) analog for aperiod of at least two months upon administration, which comprisespreparing a water-in-oil emulsion comprising an inner aqueous phase freefrom a drug retaining substance containing 35 to 60% (W/W) of the LH-RHanalog, and an oil phase containing a homopolymer of lactic acid or acopolymer of lactic acid and glycolic acid with a ratio of lacticacid/glycolic acid of 100/0 to 90/10, the homo-or copolymer having aweight-average molecular weight of 13,800 to 19,000 and a dispersiondegree (ratio of weight-average molecular weight to number averagemolecular weight) of 1.5 to 2.5, as the material for forming an outerwall of the microcapsule, and then subjecting said water-in-oil emulsionto in-water drying or phase-separation.
 6. A process according to claim5, wherein the water-in-oil emulsion is dispersed in an aqueous phaseand the resulting water/oil/water ternary emulsion is subjected toin-water drying.
 7. A process according to claim 5, wherein thewater-in-oil emulsion is dispersed in an aqueous phase containingpolyvinyl alcohol as an emulsifying agent.
 8. A process according toclaim 5, wherein the LH-RH analog is water-soluble and has a molecularweight of 1,000 or more.
 9. A process according to claim 5, wherein theLH-RH analog is (pyr)Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHC₂ H₅.
 10. Amicrocapsule according to claim 1, wherein the oil phase contains ahomopolymer of lactic acid.
 11. A microcapsule according to claim 1,wherein the oil phase contains a homopolymer of lactic acid having aweight-average molecular weight of 14,100 to 18,200 or a lacticacid/glycolic acid copolymer with a ratio of lactic acid/glycolic acidof 100/0 to 90/10 that has a weight-average molecular weight of from13,800 to 19,000.
 12. A microcapsule according to claim 1, wherein theoil phase contains a lactic acid/glycolic acid copolymer with a ratio oflactic acid/glycolic acid of 100/0 to 90/10.
 13. A microcapsuleaccording to claim 1, wherein the oil phase contains a lacticacid/glycolic acid copolymer with a ratio of lactic acid/glycolic acidof 100/0 to 90/10 that has a weight-average molecular weight of from13,800 to 19,000.
 14. A process according to claim 5, wherein the oilphase contains a homopolymer of lactic acid.
 15. A process according toclaim 5, wherein the oil phase contains a homopolymer of lactic acidhaving a weight-average molecular weight of 14,100 to 18,200, or alactic acid/glycolic acid copolymer with a ratio of lactic acid/glycolicacid of 100/0 to 90/10 that has a weight-average molecular weight offrom 13,800 to 19,000.
 16. A process according to claim 5, wherein theoil phase contains a lactic acid/glycolic acid copolymer with a ratio oflactic acid/glycolic acid of 100/0 to 90/10.
 17. A process according toclaim 5, wherein the oil phase contains a lactic acid/glycolic acidcopolymer with a ratio of lactic acid/glycolic acid of 100/0 to 90/10that has a weight-average molecular weight of from 13,800 to 19,000.